Caged ligands and uses thereof

ABSTRACT

Provided are caged compounds comprising a ligand that specifically reacts with a receptor not naturally present in mammals. The cage is released from the ligand upon illumination of the compound with light. Also provided are cells transfected with a gene of interest and a gene encoding a receptor, the gene of interest operably linked to a genetic element capable of being induced by the receptor when bound to a ligand, and the receptor not naturally present in the species of the cell. The cells also comprise a caged ligand of the receptor. Additionally provided are methods of inducing a gene of interest in the above cells. Also provided are methods of repressing a gene of interest in a cell using caged ligands of receptors. Methods are additionally provided for inducing elimination of a target sequence in a cell of a species, using a caged ligand and a recombinase.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/420,898, filed Oct. 24, 2002.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of GrantsNo.R21GM068993 and 7R01CA075503, both awarded by the National Institutesof Health.

BACKGROUND

(1) Field of the Invention

The present invention generally relates to regulation of geneexpression. More specifically, the invention relates to regulation ofgene expression using caged ligands that bind and activate receptorswhen uncaged by exposure to light.

(2) Description of the Related Art

REFERENCES CITED

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Knock-out and knock-in animal models are commonly employed to assess thebiological role of specific proteins in the context of a multicellularorganism. However, expression of individual genes is a temporal- andspatial (i.e. tissue)-specific phenomenon that can influence both normaland abnormal biological processes. For example, it has not been possibleto study the role of certain genes (cyclin D1, Stat5A, prolactinreceptor) in mammary gland tumorigenesis because transgenic mice missingthese genes lack proper mammary gland development (Sinn et al., 1987;Ormandy et al., 1997; Liu et al., 1997). Indeed, knockouts fail toreproduce a key element that drives carcinogenesis in general, namelythe acquisition of somatic mutations in the adult.

To overcome these limitations, systems have been engineered thatinducibly regulate the transgene of interest or excise the targeted geneof choice (Ryding et al., 2001; Albanese et al., 2002). These inducibleconstructs allow gene expression patterns to be temporally controlledduring growth and development as well as at any point during thelifespan of the animal. The characteristics of an ideal inducibletransgenic system include low basal level expression and robustinduction of the transgene, the lack of secondary or deleterious effectsof the inducing agent, tissue specific targeting, and the ability tosustain transgene induction. These characteristics are particularlyimportant in the delivery of embryonic lethal, transforming, orotherwise toxic genes. For example, potent oncogenes such as myc exhibita wide range of biologic effects, and therefore the ability to controlboth the temporal expression profile and the activity of the gene iscritical.

The early inducible transgenic lines relied on the administration ofheavy metals or naturally occurring steroid hormones, such asglucocorticoids, to provoke transgene expression (Gingrich & Roder,1998). However, heavy metals are toxic and glucocorticoids regulate avariety of endogenous genes, thereby complicating interpretation of thebiological response to the inducing agent. A variety of additionalinducing agents have also been described recently, includingtetracycline (tet operon system) (Shockett & Schatz, 1996), IPTG (Lacoperon repressor system) (Cronin et al., 2001), FK1012 (FKBP induciblesystem) (Belshaw et al., 1996), tamoxifen (estrogen receptor system)(Feil et al., 1996), and ecdysone receptor agonists (ecdysone receptorinducible system) (No et al., 1996; Albanese et al., 2000). However,many of these inducible systems are plagued by difficulties such asmosaic induction, toxicity, background transgene expression, sluggishclearance, and poor expression of the transactivator.

In 1996, Evans and his colleagues described an ecdysone-inducible geneexpression construct (No et al., 1996). Ecdysone, the insect moltinghormone, triggers metamorphosis by binding to and activating the nuclearheterodimer of the ecdysone receptor (EcR) and the product of theultraspiracle gene (USP). The activated complex, in association with anecdysone-responsive element (EcRE), subsequently drives gene expression.In the mammalian construct, the EcR and retinoid X receptor (RXR; themammalian homologue of USP) are constitutively produced. The gene ofinterest, which is operably linked to the EcRE, is expressed uponintroduction of ecdysone (or structurally related analogs). Theadvantages of this system include low basal expression, highinducibility (up to 4 orders of magnitude), and the fact thatecdysteroids are not toxic and do not affect mammalian physiology.

Inducible gene expression systems, as they are currently devised,provide temporal control over when the gene of interest is activatedduring the lifetime of the animal. However, fine spatial control overwhere gene expression is induced is problematic. Studies to date haveutilized tissue-selective promoters, such as a modified MMTV promoterconstruct, to enhance ecdysteroid-induced transgene expression in themammary gland (Albanese et al., 2000).

Unfortunately, in a very real sense, this technology has limitations,e.g., in cancer studies, because it does not faithfully recapitulate theprocess of tumorigenesis in the adult. For example, most breast cancersare thought to arise via the oncogenic transformation of epithelialcells that line the mammary ducts followed by clonal expansion (Wazerand Band, 1999). Although tumorigenesis likely proceeds via thetransformation of specific individual cell types in anatomicallywell-defined regions, the relative tumorigenic potential of differentmammary precursor cells remains a mystery. Inducible gene expressionsystems devised to date do not offer the fine spatial control to explorethe relationship between tissue microenvironment and the pathogenesis ofvarious disease states.

For these and other reasons, there is a need for methods and geneticconstructs that enable fine spacial and temporal control of geneticregulation of genes of interest. The present invention addresses thatneed.

SUMMARY OF THE INVENTION

Accordingly, the inventors have discovered that spacial and temporalcontrol of expression of genes of interest in a cell can be achievedusing certain caged ligands of receptors, for example where the receptoris not naturally present in the species that the cell belongs.

Thus, in some embodiments, the present invention is directed tocompounds comprising a ligand that specifically reacts with a firstreceptor not naturally present in mammals. In these embodiments, thecompounds further comprise a molecular cage covalently bound to theligand that prevents reaction of the ligand with the first receptor,where the ligand in these embodiments is released from the cage andcapable of reacting with the first receptor upon exposure of thecompound to light.

In other embodiments, the invention is directed to cells of a species,where the cells are transfected with a gene of interest and a geneencoding a first receptor, the gene of interest operably linked to agenetic element capable of being induced by the first receptor whenbound to a ligand, and the first receptor not naturally present in thespecies. In these embodiments, the cells further comprise a compoundcomprising the ligand and a molecular cage covalently bound to theligand that prevents reaction of the ligand with the first receptor,where the ligand is released from the cage and capable of reacting withthe first receptor upon exposure of the compound to light.

The invention is also directed to methods of expressing a gene ofinterest in a cell of a species. The methods comprise creating theabove-described cells of a species by transfecting the cell with thegene of interest and a gene encoding a first receptor, the gene ofinterest operably linked to a genetic element capable of being inducedby the first receptor when bound to a ligand, the first receptor notnaturally present in the species; and adding a compound to the cell, thecompound comprising the ligand and a molecular cage covalently bound tothe ligand that prevents reaction of the ligand with the first receptor,the ligand capable of being released from the cage upon exposure of thecompound to light; then exposing the cells of a species to lightsufficient to release the cage from the ligand.

The present invention is additionally directed to methods of expressinga second gene of interest in a cell of a species. The methods comprisetransfecting the cell with a first gene of interest and a gene encodinga first receptor, where the first gene of interest encodes a viralreceptor, the viral receptor allowing entry of a viral vector into thecell. In these embodiments, the first gene of interest is operablylinked to a genetic element capable of being induced by the firstreceptor when bound to a ligand and the first receptor is not naturallypresent in the species. The ligand in these embodiments furthercomprises a molecular cage covalently bound to the ligand that preventsreaction of the ligand with the first receptor, where the ligand isreleased from the cage and capable of reacting with the first receptorupon exposure of the compound to light. The cell is exposed to the viralvector further comprising an expressible gene encoding the second geneof interest; then the cell is exposed to light sufficient to release thecage from the ligand, allowing the ligand to react with the firstreceptor, which directs expression of the viral receptor and allowsinfection of the cell by the viral vector; the second gene of interestis then expressed.

Additionally, the invention is directed to methods of repressing a geneof interest in a cell of a species. The methods comprise transfectingthe cell with the gene of interest and a gene encoding a first receptor,the gene of interest operably linked to a genetic element capable ofbeing repressed by the first receptor when bound to a ligand; adding acompound to the cell, the compound comprising the ligand and a molecularcage covalently bound to the ligand that prevents reaction of the ligandwith the first receptor, the ligand capable of being released from thecage upon exposure of the compound to light; then exposing the cell tolight sufficient to release the cage from the ligand.

In additional embodiments, the invention is directed to methods ofinducing elimination of a target sequence in a cell of a species. Themethods comprise transfecting the cell with: a gene encoding arecombinase operably linked to a genetic element capable of beinginduced by a first receptor when bound to a ligand, where the firstreceptor is capable of inducing the genetic element when the firstreceptor reacts with a ligand; and a gene encoding the first receptor.The methods further comprise adding a compound to the cell, the compoundcomprising the ligand and a molecular cage covalently bound to theligand that prevents reaction of the ligand with the first receptor,where the ligand is capable of being released from the cage uponexposure of the compound to light; and exposing the cell to lightsufficient to release the cage from the ligand.

The invention is further directed to kits for the conditional expressionof a gene of interest in a cell. The kits comprise, in suitablecontainers, the compound described above, comprising a ligand thatspecifically reacts with a first receptor not naturally present inmammals, where the compound further comprises a molecular cagecovalently bound to the ligand that prevents reaction of the ligand withthe first receptor, wherein the ligand is released from the cage andcapable of reacting with the first receptor upon exposure of thecompound to light. The kits also comprise a vector comprising a geneencoding the first receptor.

In related embodiments, the invention is also directed to kits for theconditional elimination of a target sequence in a cell. The kitscomprise, in suitable containers, one or more vectors comprising a geneencoding a recombinase operably linked to a genetic element capable ofbeing induced by a first receptor when bound to a ligand, where thefirst receptor is capable of inducing the genetic element when the firstreceptor reacts with a ligand. The kits also comprise a gene encodingthe first receptor, and a compound comprising the ligand and a molecularcage covalently bound to the ligand that prevents reaction of the ligandwith the first receptor, where the ligand is released from the cage andcapable of reacting with the first receptor upon exposure of thecompound to light.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a schematic depicting a method for synthesis of caged β-ecdysone4 from β-ecdysone 1.

FIG. 2 shows NMR spectra described in the Example. Panel (a) shows the1-D ¹H NMR spectrum of β-ecdysone 1 (upper) and caged β-ecdysone 4(lower). The alkylation induced change in the chemical shift of the C-3proton is highlighted. Panel (b) shows the HMBC 2-D spectrum of cagedβ-ecdysone 4. Long range coupling between the benzylic methylene protons(CH₂) of the dimethoxy-nitrobenzyl substituent and the C-2 carbon ispresent. By contrast, no such coupling is observed between CH₂ and theC-3 carbon.

FIG. 3 is a line graph of experimental results showing the generation ofluciferase activity as a function of time following photolysis. 293Tcells that had been transiently transfected with plasmids encodingconstitutively expressed EcR and RXR and inducibly expressed luciferasewere exposed to caged β-ecdysone 4 for 16 hr and then either illuminated(♦) or first washed to remove extracellular 4 and then illuminated (▴).Control luciferase expression in the absence of ligand is shown as well(▪). Percent maximal expression is normalized relative to the yieldobtained for the 1 min photolysis time period.

FIG. 4 shows micrographs of experimental results described in theExample. Panel (a) shows transfected 293T cells after exposure toβ-ecdysone 1 and subsequent probing for luciferase expression. Panel (b)shows transfected 293T cells after exposure to 4, spot illumination(˜0.25 mm²) for 10 sec, and then probing for luciferase expression.Panel (c) shows the transfected 293T cells in the experiment asdescribed in (b), outside of the region of illumination. The microscopeused in these experiments was an Olympus IX-70 @10X, N.A. 0.3.

FIG. 5 shows the chemical structures of the P-ecdysone homologuesmuristerone 6 and ponasterone A 7.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations: COSY, correlation spectroscopy; DQF-COSY, double quantumfiltered COSY; EcR, ecdysone receptor; EcRE, ecdysone response element;HMQC, heteronuclear multiple quantum COSY; HSQC, heteronuclear singlequantum COSY; RXR, mammalian homologue of USP; USP, ultraspiracleprotein.

The present invention is based on the discovery that spacial andtemporal control of expression of genes of interest in a cell can beachieved using certain caged ligands of receptors, for example where thereceptor is not naturally present in the species that the cell belongs.This discovery enables the use of various compositions and methods toprecisely control expression of genes in cells illuminated by light.

Thus, in some embodiments, the invention is directed to compoundscomprising a ligand that specifically reacts with a first receptor notnaturally present in mammals. In these embodiments, the compound furthercomprises a molecular cage covalently bound to the ligand that preventsreaction of the ligand with the first receptor, where the ligand isreleased from the cage and capable of reacting with the first receptorupon exposure of the compound to light.

These compounds are particularly useful for regulating expression of agene of interest in mammalian cells, including whole animals. Since thefirst receptor is not naturally present in mammals, expression of thegene of interest by activating the first receptor would not affect otherphysiological processes in the mammal, as would occur using a naturallyoccurring first receptor, such as the system described in Cruz et al.(2000). An example of a first receptor not present in mammals is theecdysone receptor.

As used herein, the term “reaction”, “interaction” or “binding”, whenreferring to a ligand-receptor complex, does not connote any particularbinding affinity or avidity. It only requires a specific interactionbetween the ligand and receptor that can cause a change in geneticregulation.

These embodiments are not narrowly limited to ligands that interact withany particular first receptor. They only require that theligand-receptor interaction results in a change in genetic regulation.For example, the ligand-receptor interaction could result in activationof a factor such as a response element or promoter that controlsexpression of a gene operably linked to that factor. A non-limitingexample of such a ligand-receptor system is the ecdysone-ecdysonereceptor-RXR system previously discussed. Alternatively, theligand-receptor interaction could result in down-regulation ofexpression of a gene, as occurs, for example with a nuclear co-repressor(Chen & Evans, 1995; Wang et at., 1998). Furthermore, the ligand couldbe an inhibitor of the first receptor such that its binding inhibitsactivation of the first receptor by an activating ligand.

In preferred embodiments, the ligand is a small molecule, less than 1000Dalton. However, the invention also encompasses ligands that are aminoacids, oligopeptides, polypeptides, saccharides, lipids, nucleotides,nucleic acids, metal ions, etc. provided the ligand is capable ofactivating a first receptor not naturally present in mammals. The mostpreferred ligands are those that, when caged, are capable of passinginto a target cell without excessive manipulation such aselectroporation or liposome encapsulation.

In some preferred embodiments, the ligand interacts with an ecdysonereceptor. The ligand-ecdysone receptor combination is particularlyuseful in these embodiments because the ecdysone-responsive element(EcRE) allows low basal expression in the absence of the ligand-ecdysonereceptor combination, and provides for high induction of the gene ofinterest when the ligand-ecdysone receptor is present. Additionally, theligands generally are not toxic and do not affect mammalian physiology.Furthermore, there are many ecdysone receptor activating ligands ofdisparate structure, allowing for utilization of numerous cagingchemistries. Most of the ecdysone receptor ligands are steroids, butothers are not. See, e.g., Dinan et al., 1999; Saez et al., 2000;Mikitani, 1996; U.S. Pat. No. 6,258,603. Non-limiting examples includeecdysone, 20-hydroxyecdysone, ponasterone A, muristerone A,inokosterone, 3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide anddibenzoylhydrazines such as tebufenozide and1,2-dibenzoyl-1-tert-butyl-hydrazine. The skilled artisan would beexpected to be able to create an inactive caged version of essentiallyany ecdysone receptor ligand without undue experimentation.

The invention encompasses the use of any appropriate photolysingmolecular cage, now known or later discovered. It is contemplated thatthe skilled artisan could choose the cage without undue experimentationaccording to the needs for the individual application. Many differentcages are known in the art, and several have been applied to biologicalmolecules. See, e.g., Adams & Tsien, 1993; Curley & Lawrence, 1999;Marriott & Walker, 1999; Furuta et al., 1999; U.S. Pat. No. 5,635,608.These include single photon cages (susceptible to wavelengths of between300-400 nm, more preferably 325-375 nm, even more preferably 340-360 nm;most preferably about 350 nm—see Example) and two-photon cages(generally susceptible to higher wavelengths, e.g., about 700 nm). Thegreater efficiency of the latter cages and their susceptibility tohigher wavelengths of light that more readily penetrates tissues thansingle photon cages makes two-photon cages particularly useful whereactivation of genes of interest is desired in cells that are not on atissue surface. Nonlimiting examples of two-photon cages includebrominated 7-hydrozxycoumarin-4-ylmethyls. See, e.g., Furuta et al.,1999; Pettit et al., 1997.

When the ligand is a steroid, such as a steroid that is an ecdysonereceptor ligand (e.g., ecdysone—see Example), a preferred molecular cageis a nitromethoxybenzyl moiety, more preferably a1-methyl-4,5-dimethoxy-2-nitrobenzene (see FIG. 1).

In other embodiments, the invention is directed to cells of a speciesthat are transfected with a gene of interest and a gene encoding a firstreceptor, where the gene of interest is operably linked to a geneticelement capable of being induced by the first receptor when bound to aligand, and where the first receptor is not naturally present in thespecies. In these embodiments, the cells further comprise a compoundcomprising the ligand and a molecular cage covalently bound to theligand that prevents reaction of the ligand with the first receptor,wherein the ligand is released from the cage and capable of reactingwith the first receptor upon exposure of the compound to light.

As used herein, the term “genetic element” is to be broadly construed asany nucleic acid sequence in the genome of the species that, wheninteracting with the ligand-receptor, is induced, affectingtranscription of the gene of interest. Included are promoters,enhancers, suppressing elements, etc. In this definition, the geneticelement is preferably 5′, and in close proximity to the gene ofinterest, such as a promoter. However, the invention encompasses geneticelements that are not physically linked to the gene of interest.

The cells in these embodiments can be of any prokaryotic or eukaryoticspecies, such as animal cells or plant cells. Because of theapplications to human medicine, the animal cells are preferablymammalian cells, most preferably human cells. Preferably, the cells arepart of a multicellular organism, since a particular advantage of theinvention is the ability to induce the gene of interest in a particulargroup of cells in a tissue, for example cancer cells, e.g., in a tumor.However, cells in culture, either prokaryotic or eukaryotic cells, arealso envisioned as within the scope of the invention. As such, the cellsare useful when very synchronous induction of the gene of interest isrequired, since induction by light is much more synchronous thaninduction by adding the (uncaged) ligand to a culture because the lightinducing system only requires the photolysis of the cage and not thediffusion of the ligand into the cell. The cells of the invention arealso useful with cells in culture when induction of the gene of interestis desired in only some of the cells.

In these embodiments, where the cell is part of a multicellularorganism, the transfected cells could include one cell or a few cells.However, since spacial control of regulation of the gene of interest iscontemplated to be by shining the photolysing wavelengths of light ontothe appropriate cells, there does not need to be concern that cells aretransfected where the activation of the gene of interest is not desired.Thus, substantially all of the cells of a cell type in the organism, orall the cells of the organism can be transfected with the gene ofinterest and the first receptor.

It is preferred that the receptor-ligand-genetic element system chosenfor these embodiments have minimal basal transcription level of the geneof interest, particularly when expression of the gene of interest innon-target cells is detrimental. In most embodiments, it is alsopreferred that the difference between the basal transcription level andthe induced transcription level is great, preferably at least double,more preferably at least five-fold, even more preferably at leastten-fold, and most preferably at least fifty-fold.

Any receptor-ligand-genetic element system not naturally present in thespecies can be used in these embodiments. Selection of such a system iswithin the skill of the art, without undue experimentation. In speciesnot having the ecdysone receptor, e.g., mammals or bacteria, theecdysone/ecdysone receptor/ecdysone-response element is generallypreferred.

In some preferred embodiments, particularly when the cell is avertebrate (e.g., mammalian) cell, the compound is one of the previouslydescribed compounds. However, the invention is not limited to thosecompounds that comprise ligands to receptors not naturally present inmammals. The skilled artisan would understand that the invention wouldbe useful for controlling expression of genes of interest in cells ofvirtually any species, with any first receptor that inducestranscription upon interaction with a ligand.

The genetic element capable of being induced by the first receptor ispreferably not inducible by another receptor that could be present inthe transfected cell. To prevent side effects of the light induction(e.g., altered transcription of genes that are not the gene ofinterest), it is also preferred that the genetic element is not alsonaturally present in the cell that is exposed to the photolysing light.The genetic element can also be naturally occurring or geneticallymanipulated, e.g., to affect the basal or induced transcription rates.

In embodiments where the first receptor requires two or more proteins,such as the ecdysone receptor, genes for both proteins could be providedtransgenically. Such a case is useful, e.g., if the RXR/USP component ofthe ecdysone receptor is not naturally present in the cell, is presentin limiting amounts, or unexpectedly poorly interacts with the EcR andligand.

Where the ecdysone receptor and an ecdysone receptor ligand is used, anecdysone receptor from any species having such receptors can be used.The skilled artisan could identify a previously unidentified ecdysonereceptor gene, and isolate and genetically manipulate that gene withoutundue experimentation using known methods and present knowledge aboutother ecdysone receptor genes.

The gene of interest that can be transfected into the cells of thepresent invention is not limited to any particular type of gene, andincludes both genes that can be translated (into oligopeptides orpolypeptides) as well as useful genes that encode an untranslated RNAsuch as an antisense gene, an aptamer, or an siRNA, of which there aremyriad examples known in the art. Nonlimiting examples of translatablegenes that could be useful in the present invention include genesencoding apoptosis-inducing proteins, proteins comprising an antibodybinding domain, angiogenic factors, cytokines, viral receptors, bloodproteins, transcription factors, structural proteins, viral proteins,bacterial proteins, other extracellular proteins, proteins alreadypresent in the cell (for the purpose of, e.g., overexpressing theprotein), and engineered proteins with no natural counterpart.

In some embodiments, the gene of interest encodes a recombinase suchthat, upon light induction, the recombinase is synthesized, causingelimination of a target sequence. The recombinase can be any recombinasenow known or later discovered that could be used for this purpose.Nonlimiting examples include Cre recombinase, flp recombinase, Intrecombinase, TpnI and β-lactamase transposons, Tn3 resolvase, SpoIVCrecombinase, Hin recombinase, and Cin recombinase. See, e.g., discussionin U.S. Patent Application Publication 20020152493 at paragraph 66, andthe references cited therein, all incorporated by reference. A preferredrecombinase is Cre recombinase, because of its precision and ease of use(Nagy, 2000). Where the recombinase is a Cre recombinase, the targetsequence is flanked by two loxP sites (Id). As used herein, a loxP siteis a sequence consisting of two 13 bp inverted binding sites separatedby a 8 bp spacer. The wild-type loxP sequence(ATAACTTCGTATAATGTATGCTATACGAAGTTAT) can be used, or any known loxPmutants, such as those which are designed to prevent the presence of aresidual loxP site after recombination (see, e.g., Arakawa et al.,2001).

In the embodiments where the gene of interest is a recombinase, theinvention is not limited to any particular target sequence to beeliminated. Nonlimiting examples of a useful target sequence include atarget sequence comprising a promoter, such that elimination of thetarget sequence prevents a gene operably linked to the target sequencefrom being transcribed, or a target sequence comprising a gene(including translatable genes and nontranslated genes, e.g., thoseencoding antisense RNA, aptamers, or siRNA) which would be eliminatedupon exposure to the photolysing light. Thus, the constructs where thegene of interest is a recombinase can be used to permanently eliminatethe expression of a particular gene, only in the cells exposed to thephotolysing light wavelengths.

In alternative nonlimiting embodiments, the target sequence is 3′ from apromoter, such that when the target sequence is eliminated by therecombinase, the promoter becomes operably linked to a second gene ofinterest, thus inducing the second gene of interest upon exposure of thecell to the photolysing light. In these embodiments, the second gene ofinterest can be any gene, including a gene encoding an untranslated RNAor a translatable gene. Thus, the recombinase gene of interest can beused to permanently induce expression of a second gene of interest. Thisis opposed to embodiments where the gene of interest is not arecombinase, since the induction by the uncaged ligand usually onlytemporarily induces expression of the gene of interest. See, e.g.,Example 1, where expression induced by the ecdysone receptor had ahalf-life of about 16 hours.

The embodiments where the gene of interest is a recombinase that causesoperable linkage of a promoter with a second gene of interest is notlimited to any particular type of promoter, and could include anyinducible or a constitutive promoter, as needed.

Another non-limiting example of a construct allowing permanent inductionof the second gene of interest is one where the target sequencecomprises a stop codon, such that light induction of the recombinaseeliminates the stop codon and puts the coding region of the gene inframe, allowing transcription of the complete protein encoded by thesecond gene of interest.

In embodiments where the gene of interest is a viral receptor, preferredviral receptors are viral receptors that allow entry of a viral vectorinto the cell. In so doing, a second gene of interest can be provided bythe viral vector that has been engineered to further comprise the secondgene of interest. By this method, any second gene of interest can beconveniently expressed in the illuminated cells by providing the viralvector comprising the desired second gene of interest. This methodrequires only one target organism, transfected with the first receptorand the viral receptor operably linked to a genetic element capable ofbeing induced by the first receptor when bound to a ligand, and, sinceany gene of interest can be easily expressed in the cells of theorganism by (a) adding the caged ligand and the viral vector comprisingthe gene of interest, and (b) illuminating the targeted cells. Upon suchillumination, the ligand is uncaged, allowing its binding to the firstreceptor, which then allows expression of the viral receptor. Expressionof the viral receptor then allows entry of the virus into thoseilluminated cells, and expression of the gene of interest from the viralvector. Thus, the gene of interest engineered into the viral vector isonly expressed in the illuminated cells. A nonlimiting example of such aviral vector and viral receptor is the TVA receptor and subgroup A avianleucosis virus, for example comprising an RCAS vector. See Orsulic etal., 2002; Fisher et al., 1999; Pao et al., 2003.

The above cells, already exposed to light and the viral vectorexpressing the second gene of interest, where the viral vector hasinfected the cell and expresses the second gene of interest, are alsocontemplated as being within the scope of the invention.

In these embodiments, the second gene of interest is not limited to anyparticular gene. As with previous embodiments, preferred second genes ofinterest encode apoptosis-inducing proteins, proteins comprising anapoptosis-inducing protein, proteins comprising an antibody bindingdomain, angiogenic factors, cytokines, blood proteins, transcriptionfactors, structural proteins, viral proteins, bacterial proteins,extracellular proteins, proteins already present in the cell, andengineered proteins with no natural counterpart. The second gene ofinterest can also encode an untranslated RNA, for example an antisenseRNA, an aptamer, and an siRNA.

The invention is not limited to any particular method of transfectingthe cells with the gene of interest and the gene encoding a firstreceptor. The gene of interest could be on the same or on differentvectors as the gene encoding the first receptor. The vectors used canalso be any vector appropriate for the cell being transfected, andinclude viral vectors and naked DNA vectors such as plasmids. Theskilled artisan would generally be expected to determine, without undueexperimentation, suitable transfection vectors and methods for anyparticular cell.

The invention also encompasses the use of transfection methods andvectors that allow stable as well as transient expression of the firstreceptor; extrachromosomal as well as chromosomally integrated (eitherby homologous or heterologous recombination) transfection of the cell isalso envisioned. The cells of the invention could also be descended froma transfected cell, for example a cell from a transgenic organism whosegerm line was transfected with the gene of interest and/or the geneencoding a first receptor; or the progeny of such an organism.

The compound can enter the cell by any means appropriate, as could bedetermined by the skilled artisan without undue experimentation. Itwould be expected that the compound might degrade or otherwise becomeinactive in the cell over time. The skilled artisan would understandthat the rate of such degradation would depend on the chemical stabilityof the compound as well as the presence in the cell of enzymes thatcould degrade the compound.

The present invention is also directed to methods of inducing a gene ofinterest in a cell of a species. The methods comprise, first, creatingany of the above described cells. The cells are created in two steps,which could be performed in any order. One step is transfecting the cellwith the gene of interest and a gene encoding a first receptor, wherethe gene of interest is operably linked to a genetic element capable ofbeing induced by the first receptor when bound to a ligand, and thefirst receptor is not naturally present in the species. The other stepis adding a compound to the cell, where the compound comprises theligand and a molecular cage covalently bound to the ligand that preventsreaction of the ligand with the first receptor, the ligand capable ofbeing released from the cage upon exposure of the compound to light. Thecell is then exposed to light sufficient to release the cage from theligand.

In preferred embodiments, the cell in these methods is part of a livingmulticellular organism, where some or all of the cells can betransfected with the gene of interest and the first receptor.

The light used to release the cage from the ligand must includewavelengths appropriate and of sufficient intensity to cause uncaging ofthe cage from the ligand. For example, where the cage is a single photoncage, the light preferably comprises wavelengths at 300-400 nm; morepreferably at 325-375 nm. Alternatively, where the cage is a two-photoncage, the light generally must comprise wavelengths of about 600-800 nm,more preferably 650-750 nm, most preferably about 700 nm.

As previously discussed in the context of the cells of the invention,the transfection of the cell can be by any appropriate means, using anyappropriate vector type, etc. Additionally, these methods include theuse of transgenic cells descended from a transfected cell, for example acell from a transgenic organism whose germ line was transfected with thegene of interest and/or the gene encoding a first receptor; or theprogeny of such an organism. Other parameters previously discussed inthe context of the cells of the invention also applies to the cellsuseful for these methods.

The skilled artisan would understand that these methods are particularlyuseful for inducing a gene in a cell or cells at a precise location,e.g., in a tissue such as cancerous tissue in a mammal, for example ahuman cancer patient. Since the first receptor is not normally presentin the species, the activation of the first receptor by uncaging thecompound would not be expected to cause altered transcription other thanof the gene of interest.

Where the gene of interest is a recombinase such as a Cre recombinase,as previously described in the context of the cells of the invention, atarget sequence is permanently eliminated only from the cells exposed tophotolysing light. Depending on the design of the genetic constructtransfected into the cell, the elimination of the target sequence canhave the effect of permanently eliminating expression of a second geneof interest (e.g., if the target sequence encodes the gene or a geneticelement for the gene), inducing expression of a second gene of interest(e.g., if eliminating the target sequence eliminates a stop codon,preventing translation of the complete second gene of interest), orpermanently altering expression of the second gene of interest (e.g., ifeliminating the target sequence eliminates one promoter operably linkedto the second gene of interest and operably links the second gene ofinterest with a second promoter with different expression parametersthan the first promoter). These methods are entirely within the skill ofthe art. Furthermore, a skilled artisan could design other constructswith useful effects as necessary for any particular application.

The present invention is additionally directed to methods of expressinga second gene of interest in a cell of a species, utilizing a viralvector-viral receptor expression system. The methods comprisetransfecting the cell with a first gene of interest and a gene encodinga first receptor, where the first gene of interest encodes a viralreceptor, the viral receptor allowing entry of a viral vector into thecell. In these embodiments, the first gene of interest is operablylinked to a genetic element capable of being induced by the firstreceptor when bound to a ligand and the first receptor is not naturallypresent in the species, as described above. The ligand in theseembodiments further comprises a molecular cage covalently bound to theligand that prevents reaction of the ligand with the first receptor,where the ligand is released from the cage and capable of reacting withthe first receptor upon exposure of the compound to light as describedabove. The cell is exposed to the viral vector further comprising anexpressible gene encoding the second gene of interest. The cell is thenexposed to light sufficient to release the cage from the ligand,allowing the ligand to react with the first receptor, which directsexpression of the viral receptor and allows infection of the cell by theviral vector. The second gene of interest is then expressed.

A preferred example of the viral receptor-viral vector system utilizesthe TVA receptor for subgroup A avian leucosis virus, and the viralvector is a subgroup A avian leucosis virus, as described in the contextof the cells of the invention, and in Orsulic et al., 2002, Fisher etal., 1999, and Pao et al., 2003. That system is preferred because theRCAS vector utilized therein is flexible and well developed.

A preferred first receptor is an ecdysone receptor, and some preferredligands are steroids, particularly when the first receptor is anecdysone receptor. Preferred examples of steroid ligands for theecdysone receptor are ecdysone, 20-hydroxyecdysone, ponasterone A,muristerone A, inokosterone,3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide and adibenzoylhydrazine.

As with other embodiments, the second gene of interest can be operablylinked to an inducible promoter or a constitutive promoter. Nonlimitingexamples of preferred second genes of interest are genes encoding anapoptosis-inducing protein, a protein comprising an apoptosis-inducingprotein, a protein comprising an antibody binding domain, an angiogenicfactor, a cytokine, a blood protein, a transcription factor, astructural protein, a viral protein, a bacterial protein, anextracellular protein, a protein already present in the cell, or anengineered protein with no natural counterpart. Alternatively, thesecond gene of interest can encode an untranslated RNA, for example anantisense RNA, an aptamer, or an siRNA.

The invention is additionally directed to methods of repressing a geneof interest in a cell of a species. These methods comprise transfectingthe cell with the gene of interest and a gene encoding a first receptor,the gene of interest operably linked to a genetic element capable ofbeing repressed by the first receptor when bound to a ligand; adding acompound to the cell, the compound comprising the ligand and a molecularcage covalently bound to the ligand that prevents reaction of the ligandwith the first receptor, the ligand capable of being released from thecage upon exposure of the compound to light; then exposing the cell tolight sufficient to release the cage from the ligand.

These methods are not narrowly limited to any particular repressingfirst receptor. Examples of some such useful first receptors includetranscriptional co-repressors (Chen & Evans, 1995; Wang et al., 1998).Preferably, in these embodiments, the first receptor is not naturallypresent in the species, to prevent undesirable side effects caused bythe induction.

These methods are entirely analogous with the previously describedmethods of inducing a gene of interest, except a repressing firstreceptor, rather than an inducing first receptor, is used. Thus, thecells can be prokaryotic or eukaryotic; they can also be part of aliving multicellular organism; the gene of interest can be anytranslatable or non-translatable gene, etc.

These embodiments are useful where the induction of a gene is notdesired at particular times and/or places (e.g., in particular cells ofa tissue, such as cancer cells). In some embodiments, the gene ofinterest is one that is normally present under native regulation (e.g.,under the control of a native constitutive or inducible promoter), andgene of interest and the genetic element capable of being repressed bythe first receptor is transfected into the cell by homologousrecombination. Exposing such a cell to light would thus repress thenative gene. Other applications of these methods would be readilyapparent to the skilled artisan.

In related embodiments, the invention is also directed to methods ofinducing elimination of a target sequence in a cell of a species. Thesemethods comprise transfecting the cell with: a gene encoding arecombinase operably linked to a genetic element capable of beinginduced by a first receptor when bound to a ligand, where the firstreceptor is capable of inducing the genetic element when the firstreceptor reacts with a ligand; and also transfecting the cell with agene encoding the first receptor. The methods also comprise adding acompound to the cell, the compound comprising the ligand and a molecularcage covalently bound to the ligand that prevents reaction of the ligandwith the first receptor, where the ligand is capable of being releasedfrom the cage upon exposure of the compound to light. After the abovesteps, the cell is exposed to light sufficient to release the cage fromthe ligand.

As with previously described embodiments, a preferred recombinase is Crerecombinase. In those embodiments employing a Cre recombinase, the cellmust also be transfected with two loxP sites flanking the targetsequence. The scope of the various aspects of these embodiments havebeen discussed infra, in the context of other methods and compositions.

The invention is additionally directed to kits for the conditionalexpression of a gene of interest in a cell. The kits comprise, insuitable containers, the compounds described above that comprises aligand that specifically reacts with a first receptor not naturallypresent in mammals, where the compound further comprises a molecularcage covalently bound to the ligand that prevents reaction of the ligandwith the first receptor, and where the ligand is released from the cageand capable of reacting with the first receptor upon exposure of thecompound to light. The kits also comprise a gene encoding the firstreceptor.

These kits can comprise components that are particularly useful for theviral receptor-viral vector system described above. For example, thefirst vector of the kit can comprise a gene encoding a viral receptor,where the viral receptor allowing entry of a viral vector into a cell,and the kit can also comprise the viral vector comprising a site forinsertion of the gene of interest such that the gene of interest can beexpressed when the viral vector infects the cell. As discussed above inthe context of the cells of the invention, a preferred viralreceptor-viral vector combination is a TVA receptor for subgroup A avianleucosis virus and a subgroup A avian leucosis virus vector. In theseembodiments, the gene of interest can be operably linked to an induciblepromoter or a constitutive promoter.

The kits can be directed to be used with any prokaryotic, eukaryotic orarchaeal cell. In some preferred embodiments, the cell is a mammaliancell. It is also preferred that the cell is part of a multicellularorganism.

The kits can optionally comprise instructions for expressing the gene ofinterest in the cell transfected with the vector when the cell isexposed to the compound and light.

Also within the scope of the invention are kits that facilitateexecuting the methods of eliminating a target sequence in a celldescribed above. These kits comprise, in suitable containers, one ormore vectors comprising (a) a gene encoding a recombinase operablylinked to a genetic element capable of being induced by a first receptorwhen bound to a ligand, where the first receptor is capable of inducingthe genetic element when the first receptor reacts with a ligand; and(b) a gene encoding the first receptor. The kits also comprise acompound comprising the ligand and a molecular cage covalently bound tothe ligand that prevents reaction of the ligand with the first receptor,where the ligand is released from the cage and capable of reacting withthe first receptor upon exposure of the compound to light.

As discussed above in the context of the methods of eliminating a targetsequence, a preferred recombinase is a Cre recombinase. Also, the firstreceptor is preferably an ecdysone receptor, and some preferred ligandsare steroids, particularly when the first receptor is an ecdysonereceptor. Preferred examples of steroid ligands for the ecdysonereceptor are ecdysone, 20-hydroxyecdysone, ponasterone A, muristerone A,inokosterone, 3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide and adibenzoylhydrazine. These kits also optionally comprise instructions forusing the kit to eliminate a target sequence in cells transfected withthe vectors and exposed to the compound and to light.

Preferred embodiments of the invention are described in the followingexample. Other embodiments within the scope of the claims herein will beapparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples.

EXAMPLE Spacial and Temporal Gene Regulation using Caged β-EcdysoneExample Summary

Transgene-based inducible expression systems furnish the means to studythe influence of any gene of interest at any point during an organism'slifetime. This powerful technology adds a temporal dimension to theexisting knockout strategies commonly employed to assess proteinfunction in vivo. However, in biological systems, the expression ofindividual genes is both temporally and spatially (i.e.cell/tissue)-regulated. Consequently, an experimental methodology thatfurnishes both temporal and spatial control over transgene expressionwould provide the additional spatial dimension to assess proteinfunction in the context of tissue microenvironment. We describe hereinthe creation and study of a light-activatable counterpart to thepreviously reported ecdysone-inducible gene expression system. Astannylene acetal-based strategy was employed to prepare the firstexample of a caged ecdysteroid. The latter is nearly inactive as aninducing agent in a luciferase-based gene expression system. However,upon exposure to brief illumination, the caged ecdysteroid is rapidlyconverted into active β-ecdysone. We have found that the cagedβ-ecdysone is cell permeable, can be intracellularly photouncaged and,in combination with spot illumination, can be used to drive spatiallydiscrete protein expression in a multicellular setting. Thus, wedescribe herein a light-activatable form of ecdysone and its applicationto the spatial regulation of gene expression.

Materials and Methods

General. β-ecdysone was purchased from A. G. Scientific. All otherreagents and solvents were purchased from Aldrich. Silica gel 60 (40 μm,Baker) was employed for column chromatography. 1-D NMR spectra wererecorded on a Bruker DRX-300 and 2-D spectra on a Bruker DRX-600.Chemical shifts are reported downfield from tetramethylsilane.

Preparation of Caged β-ecdysone 4. See FIG. 1. A suspension ofβ-ecdysone (20 mg, 41.6 μmol) and dibutyltin oxide (13.5 mg, 54.2 μmol)in anhydrous methanol (5 mL) was heated to reflux for 3 hr under argon.After the solvent was removed under reduced pressure, the residue wassubsequently azeotroped with anhydrous benzene (3×2 mL). The resultingstannylene acetal was further dried in vacuo for 2 hr before addition of3 Å molecular sieves (100 mg), CsF (25.2 mg, 166.4 μmol),1-bromomethyl-4,5-dimethoxy-2-nitrobenzene 3 (20.6 mg, 74.9 μmol), andanhydrous DMF (1 mL). After the reaction mixture was stirred at roomtemperature overnight, the solvent was evaporated under reducedpressure. The resulting residue was purified by silica gel columnchromatography (methylene chloride/methanol: 6/1) to afford 4 as anoff-white solid (25.3 mg, 90%). The purity was determined to be >99% byanalytical HPLC (retention time 16.2 min on a Vydac C4 column 250 mm×3.0mm; monitored at 242 nm: a 15 min linear gradient from 95% A (water) to50% B (acetonitrile) followed by 50% B for 5 min with the flow rate of 1mL/min). ¹H-NMR (300 MHz, CD3OD): δ 0.89 (s, 3H), 0.99 (s, 3H), 1.19 (s,9H), 1.20-1.98 (m, 17H), 2.39 (m, 2H), 3.11 (m, 1H), 3.72 (m, 1H), 3.90(s, 3H), 3.97 (s, 3H), 4.25 (br s, 1H), 4.99 (q, 2H), 5.81 (d, 1H), 7.42(s, 1H), 7.71 (s, 1H). HRMS (ESI+) Calcd for C₃₆H₅₄NO₁₁: 676.3697 Found:676.3674. A series of 2-D correlation (COSY) NMR experiments weresubsequently performed to assess the site of alkylation on theβ-ecdysone framework, including double quantum filtered COSY (DQF-COSY),heteronuclear single quantum correlation (HSQC), and heteronuclearmultiple quantum correlation (HMQC) spectroscopies.

Photolysis of Caged β-ecdysone 4. A 500 μL 100 μM solution of 4 in 2%methanol/98% water was placed in a 24-well culture plate. Photolysis wasperformed using an Oriel 200 W Hg arc lamp (model 6283) with a 348 nmfilter (Oriel, lot number 51260; 50% internal transmittance at 348 nmand cutoff at 325 nm) to remove short wavelength light and an IR filterto remove heat. Aliquots of 10 μL were removed at different time pointsof photolysis and were analyzed by analytical HPLC (monitored at 242 nm)employing a Vydac C4 (250 mm×3.0 mm) column and using the followingsolvent system: a linear gradient from 95% A (water):5% B (acetonitrile)to 50% A (water):50% (acetonitrile) over 15 min followed by 50% A(water):50% B (acetonitrile) for 5 min with flow rate at 1 mL/min.

Plasmid Description. The luciferase reporter (E/GRE)₆TK81LUC consists ofmultimeric E/GRE binding sites from (E/GRE)₄ΔMTVLuc linked to theminimal TK promoter in the pA₃LUC reporter plasmid. The modified MMTVpromoter from MMTVp206 that incorporates the 5′UTR of v-Ha-ras wasinserted into a vector containing a modified ecdysone receptor VgEcR (agift from R. Evans) to form MMTV-VgEcR.

Cell Culture, DNA Transfection, and Luciferase Assays. Cell culture, DNAtransfection, and luciferase assays were performed as previouslydescribed (Wantanable et al., 1966). Briefly, 293-derived BOSC cells(293T) were seeded into individual wells of a 24 well plate andmaintained in Dulbecco's modified Eagles medium with 10% fetal calfserum and 1% penicillin/streptomycin. Cells were transiently transfectedwith pVgEcR/RXRα and (E/GRE)₆TKLUC via a standard calcium phosphatemethod. The media was changed after 14 hr and the cells treated with 10μL of 5 mM β-ecdysone 1 (FIG. 1) in methanol (to furnish a 100 μM finalconcentration of ecdysteroid). Cells were either left un-illuminated orilluminated for 1 min using an Oriel 200 W Hg arc lamp (model 6283) witha 348 nm filter (Oriel, lot number 51260, 50% internal transmittance at348 nm and cutoff at <325 nm) to remove short wavelength light and twoIR filters to remove heat. Cells were subsequently lysed as previouslydescribed and the luciferase assay performed at room temperature usingan Autolumat LB 953 (EG & G, Berthold). Luciferase content was measuredby calculating the light emitted during 10 sec of the reaction. Thevalues are expressed in arbitrary light units.

Time-dependent luciferase assays. 293T cells were split in a 24-wellculture plate (Becton Dickinson Labware, lot #353047) and weretransfected by a calcium phosphate method. The cells in 500 μL culturemedium were incubated with either 100 μM caged β-ecdysone, 100 μMβ-ecdysone, or absent an ecdysteroid ligand. After the cells wereincubated with caged β-ecdysone for 16 hours, the medium was removed andwashed with PBS once. A fresh medium free of ligand was then immediatelyadded just prior to UV light exposure for 1 min. In the “Caged+hv”experiment, the caged β-ecdysone ligand remained in the medium and thesecells were likewise illuminated for 1 min. Cells that were not exposedto ecdysteroid ligand were treated in an otherwise analogous fashion.The cells were then returned to the incubator for various timeintervals. Incubation was terminated by treating cells with anextraction buffer (1% Triton X-100 and 1 mM DTT in GME). Luciferaseactivity was preformed as described above.

Immunodetection of Intracellularly Expressed Luciferase. 293T cells weretransferred to the individual wells of a 24-well culture plate (BectonDickinson Labware, lot #353047) and transfected as described above. Thecells were then incubated with either 100 μM β-ecdysone or 100 μM cagedβ-ecdysone for 16 hr. The medium was removed and the cells washed oncewith PBS before addition of an ecdysteroid free medium. The surface ofthe pre-scored wells was spot illuminated (˜0.25 mm²) for 10 sec on alight microscope using a 100 W Hg-Arc lamp through an Olympus UAPO20X/0.40 objective. The cells were then incubated for 6 hr, fixed with4% formaldehyde in PBS for 60 min, washed with PBS 3×5 min,permeabilized with 0.1% TritonX100 for 10 min, washed with PBS 3×5 min,and blocked with 1% normal donkey serum (Jackson ImmunoResearch catalog#017-000-121) in PBS for 45 min. The cells were subsequently exposed to300 μL of anti-luciferase pAb (polyclonal goat antibody, Promega, lot#149040) at a concentration of 40 μg/mL in 1% donkey serum in PBS. Afterincubation for 2 hr at room temperature in a humidified chamber, thecells were washed with PBS 3×15 min and exposed to 300 μL of an AlexaFluor 568-labeled rabbit anti-goat IgG (20 μg/mL in 1% donkey serum/PBSfor 1 hr, Molecular Probes, catalog #A-11079). Unbound antibody wasremoved by washing with PBS 3×15 min and the cells treated with 300 μLof mounting medium (N-propyl gallate at 6 mg/mL in 1:1 glycerol:PBS).Images were taken using an Olympus IX-70 microscope equipped with a CCDcamera.

Results

Synthesis and Characterization of Caged β-Ecdysone 4. We initiallysought to prepare a biologically inactive form of β-ecdysone 1 that,upon photolysis, would furnish the active ecdysteroid. A wide variety ofecdysteroid derivatives have been reported and, in general, the presenceof free hydroxyl groups at the C-2, C-3, and C-20 positions are requiredfor biological activity (Dinan et al., 1999) (FIG. 1). Consequently, weenvisioned that modification of one (or more) of these alcohol moietieswith a photosensitive substituent should furnish an inactive ecdysteroidanalog that ultimately could be “switched-on” using high intensitylight. An alkylated, as opposed to an acylated, ecdysone would enjoy theadvantage of enhanced biological stability. However, to the best of ourknowledge, we know of no report describing the alkylation of any of thehydroxyl functionalities on ecdysone or its structurally relatedcongeners. Indeed, our initial attempts to alkylate β-ecdysone with thecaging agent 3 failed to furnish a modified ecdysteroid derivative (e.g.acetonitrile/N-methyl morpholine/3).

The acetonide of the C-2/C-3 diol has been described (Suksamrarn &Pattanaprateep, 1995) and consequently we wondered whether thecorresponding tin acetal 2 could be formed in situ. Tin-based acetalintermediates have been extensively employed in carbohydrate chemistryto furnish monosubstituted derivatives in high yield and highregioselectivity (Grindley, 1998). Although the dibutylstannylene acetal2 appeared to form with ease, attempted alkylation with 3 furnished thedesired monoalkylated product in only 12% yield. We subsequentlydiscovered that 3 is prone to decomposition in the presence of base(KHCO₃, Et₃N). Although alkylation of stannylene acetal alcohol moietiesgenerally requires fairly vigorous conditions, there have been reportsthat added nucleophiles promote the desired reaction under mildconditions (Nagashima & Ohno, 1991). Indeed, we obtained a singlemonoalkylated β-ecdysone (high resolution mass spectrometry and a singlepeak by HPLC) in 90% yield when the alkylation was conducted in thepresence of CsF. Previous studies using a variety of acylating agentssuggest that the 2-OH of ecdysteroids is by far the most reactive of thehydroxyl moieties on the ecdysteroid nucleus (Dinan et al., 1999). Inaddition, extensive studies with a multitude of cis-1,2-diol tinacetal-monosaccharide intermediates revealed that the preferred site ofalkylation proceeds (often exclusively) at the equatorial hydroxyl group(Grindley, 1998).

The 3-dimensional structure of ecdysone has been described and itsstereochemical rendering is shown adjacent to 1 in FIG. 1 (Huber &Hoppe, 1965). The methyl substituent at the ring A/ring B junction liesaxial relative to the B ring, but equatorial relative to ring A.Consequently, the C-2 and C-3 hydroxyl moieties are arrangedequatorially and axially, respectively, on ring A. Based upon theseconsiderations, we predicted that alkylation should occur predominantly,if not exclusively, at the C-2 alcohol to furnish 4. However, anexamination of the ¹H NMR spectrum, in combination with previouslyreported proton chemical shifts (Galbraith & Horn, 1969), suggested thatalkylation may have transpired at the C-3 hydroxyl position (Albanese etal., 2000). The latter conclusion arises from the obviousalkylation-induced chemical shift of the C-3 hydrogen (FIG. 2 a). Giventhe difference between the predicted (4) and the apparent products (5)(FIG. 1), we obtained the complete ¹H and ¹³C chemical shift assignmentsfor β-ecdysone using a combination of ¹H-¹H (DQF-COSY), shortrange¹H-¹³C (HSQC), and long-range ¹H-¹³C (HMBC) correlation NMRspectroscopies. The HMBC spectrum of the caged β-ecdysone 4 reveals anobvious coupling between the benzyl methylene protons of the photolabilesubstituent and the carbon at the C-2 position on the ecdysteroidnucleus (FIG. 2 b). By contrast, no such coupling is observed betweenthe benzyl methylene protons and the C-3 carbon. Therefore, we concludethat alkylation proceeds at the C-2 hydroxyl to furnish 4. The mostdramatic alkylation-induced change in the 1-D spectrum (FIG. 2 a),namely the chemical shift of the C-3 proton, is best rationalized byinvoking a deshielding effect induced by the adjacent C-2 hydroxylbenzyl substituent.

Photoconversion of Caged β-Ecdysone 4 to β-Ecdysone 1. A preliminaryassessment of the light-induced conversion of the caged analog 4 toβ-ecdysone 1 was performed using a 24-well plate system. Wells in theplate were selectively photolyzed for various time intervals using a Hgarc lamp. Aliquots from the wells were subsequently analyzed by HPLC toassess formation of 1 (data not shown). A maximal photoconversion of 60%was achieved after 1 min of photolysis. Longer periods of photolysis didnot improve the overall yield of photoconversion. The photochemicalquantum yield (φ=0.034) was determined using ferrioxalate actinometry(Hatchard & Parker, 1956) according to the equation φ=ΔP/(I×t), where tis the irradiation time, ΔP is the amount of photolabile converted, andwas determined using HPLC peak areas.

Light-Driven Luciferase Expression in Transiently Transfected 293TCells. We employed a luciferase-based expression system to examine thebiological activity of β-ecdysone and its alkylated analog 4. The 293Tcell line was transiently transfected to constitutively express EcR/RXRand inducibly express (upon exposure to β-ecdysone) luciferase. Compound4 was added to the transfected 293T cells, the cell culture subsequentlyilluminated, and luciferase activity assessed (Table 1). There is littleobservable luciferase activity with culture media alone. Exposure of thecells to the bioactive β-ecdysone 1 generates a nearly 90-fold inductionof luciferase. By contrast, the caged analog 4 furnishes a slight 6-foldinduction of activity over that of culture media alone. However, 1 minphotolysis of cells treated with 4 induces a dramatic enhancement ofluciferase formation, which is approximately 60% of the expressiondisplayed by the bioactive species 1. The latter is consistent with ourobservation that a 1 min photolysis time window converts approximately60% of the caged derivative 4 into its bioactive counterpart 1. Finally,illumination in the absence of ligand fails to induce luciferaseproduction. These experiments demonstrate that light can be used toactivate the EcR/RXR gene expression system. TABLE 1 Luciferaseexpression in 293T cells transiently transfected with plasmids that codefor constitutively expressed EcR/RXR and inducibly expressed luciferasein the presence and absence of-ecdysone 1 or its caged analogue 4. Foldluciferase Experimental Conditions induction Culture media/2% MeOH —β-ecdysone 1/culture media/2% MeOH 88 ± 9 caged β-ecdysome 4/culturemedia/2% MeOH  6.5 ± 0.1 caged β-ecdysome 4/culture media/2% MeOH/1 minhν 50 ± 4 Culture media/2% MeOH/1 min hν  0.9 ± 0.1

The time-dependence of β-ecdysone-induced luciferase expressionfollowing photolysis is shown in FIG. 3. Cells were pre-incubated withthe caged β-ecdysone 4 for 16 hr, illuminated for 1 min, and then lysedat various time points following photolysis. Maximal gene expression wasobserved at 16 hr. We also assessed whether 4 is cell permeable andundergoes intracellular uncaging upon photolysis. The 293T cell line waspre-incubated with 4 for 16 hr, the media subsequently removed, and thecells washed with PBS. Ecdysone-free media was then added, the cellculture incubated for various time points, and tested for luciferaseactivity. The gene expression kinetic profile is nearly identical forthe two sets of experiments [(4+photolysis) versus(4+washing+photolysis)] during the first 5 hr, which is consistent withthe notion that caged β-ecdysone 4 is intracellularly liberated (FIG.3). However, we do note that (4+washing+photolysis) conditions achieveonly 50% of the maximal activity displayed by β-ecdysone.

Light-Driven Spatially-Discrete Luciferase Expression in TransientlyTransfected 293T Cells. We examined whether the combination of 4 andspot illumination induces gene activation in a spatially discretefashion. Transiently transfected 293T cells were incubated with 1 for 16hr, fixed, permeabilized, exposed to a luciferase antibody, and stainedwith an Alexa-labeled secondary antibody. As expected, luciferaseexpression (20% transfection efficiency) was observed throughout thegeneral cell population (FIG. 4 a). By contrast, cells incubated withthe caged analog 4 under identical conditions failed to exhibitdetectable luciferase expression (data not shown). 293T cells were alsoexposed to 4, the media replaced with fresh media to removeextracellular 4, and spot illuminated (˜0.25 mm²) for 10 sec. The cellswere then returned to the incubator for 6 hr and subsequently analyzedfor luciferase expression. Only those regions exposed to UV lightdisplay luciferase production (FIG. 4 b/4 c).

Discussion

The analysis of protein function in living animals has been andcontinues to be an exceedingly difficult endeavor. Even when functioncan be assigned to a specific protein, the phenotypic consequences ofactivation may not only be cell type specific, but can vary according toboth when (e.g. embryo versus adult) and where (e.g. specific tissuemicroenvironments) activation/expression occurs. As a result, there isincreasing interest in the development of methodologies to assessprotein function in the whole organism with respect to both temporal andspatial parameters.

Inducible gene expression systems using transgenic animals allows one toexamine the consequences of protein expression at any point during thelifetime of the animal. However, the issue of spatial control has onlyrecently begun to be addressed. One strategy is the use of light tocontrol where gene activation/protein expression transpires. Haseltonand his colleagues reported the first example of light-driven geneexpression in 1999 (Monroe et al., 1999). Those investigators prepared1-(4,5-dimethoxyl-2-nitrophenyl)diazoethane multi-modified plasmidsencoding either luciferase or green fluorescent protein. The latter weredelivered via particle bombardment (rat skin) or liposome transfection(HeLa cells). Illumination of the transfected tissue/cells produced a40-50% restoration of protein expression levels versus the control (i.e.transfection with uncaged plasmid). Recently, Okamoto and his colleaguesprepared a coumarin caged mRNA encoding various proteins, includinggreen fluorescent protein, β-galactosidase, and the transcription factorEngrailed2a, which were microinjected into Zebrafish embryos (Ando etal., 2001). An obvious advantage of the caged gene-based strategy isthat it does not require the use of transgenic animals. Furthermore, itmay ultimately be possible to simultaneously introduce and thereforeswitch on multiple genes. However, limitations include the need toresort to special delivery methods and that the mode of geneintroduction renders protein expression transient. These characteristicssuggest that the caged gene approach should prove ideal for the study ofshort-term biological phenomena in small cell populations (e.g.embryogenesis). Koh and his colleagues recently reported the preparationand use of a caged estradiol, which was used to activate the expressionof an estrogen response element-controlled luciferase gene intransiently transfected HEK293 cells (Cruz et al., 2000). This strategyoffers an exciting tool for examining the effect of hormones onendogenous gene expression profiles both in vitro and in vivo.Light-activatable, cell-permeable, small molecules possess the advantageof ready delivery throughout a large multicellular organism. Inaddition, in conjunction with the well-established Cre/loxP recombinasestrategy (Ryding et al., 2001), a single treatment could potentiallyelicit permanent changes in gene expression patterns.

We describe herein the construction and analysis of a cagedcell-permeable ecdysteroid. β-ecdysone and its structural congeners haveno known effect on mammalian physiology. Consequently, only ecdysoneresponse element-controlled transgenes should be activated uponphoto-generation of the active ecdysteroid. We also demonstrate, for thefirst time, that a small caged cell-permeable molecule can be used todrive gene expression in a spatially-discrete fashion.

Maximal biological activity of ecdysteroids requires the presence offree hydroxyl groups at the C-2, C-3, and C-20 positions (see 1) (Dinanet al., 1999). Consequently, we sought to construct a chemicallymodified biologically inactive ecdysteroid via alkylation of one ofthese alcohol moieties. Subsequent light-based cleavage of the modifyingagent from the alkylated ecdysone would then regenerate the native (andtherefore biologically active form) of ecdysone.

A wide variety of structurally diverse caging agents have been described(Adams & Tsien, 1993). We chose the 4,5-dimethoxy-2-nitrobenzyl moietyfor our initial studies because of its reasonably good photophysicalproperties (λ_(max) and Φ). Although a wide range of structurallymodified ecdysteroids have been prepared during the last four decades,we are not aware of any example in which one (or more) of the hydroxylgroups on the β-ecdysone ring system has been alkylated. Indeed, ourinitial attempts to prepare 4,5-dimethoxy-2-nitrobenzylated β-ecdysonefailed to generate any alkylated product. However, since theC-2/C-3-acetonide of β-ecdysone has been reported (Suksamrarm &Pattanaprateep, 1995), it occurred to us that it might be feasible togenerate, in situ, the dibutylstannylene acetal 3. Stannylene acetalshave been extensively employed in carbohydrate chemistry and theseintermediates are known to undergo ready alkylation in a highlyregioselective fashion (Grindley, 1998). For example, stannylene acetalsof cis-diols in 6-membered rings tend to selectively undergo alkylationon the equatorial, rather than the axial, hydroxyl moiety. With thesefeatures in mind, we exposed 2 to the bromide 3 in the presence of CsFand obtained a monalkylated product (as assessed by mass spectrometry)in 90% yield. We subsequently employed a combination of DQF-COSY, HSQC,and HMBC NMR spectroscopies to demonstrate that monoalkylation ofβ-ecdysone occurs exclusively at the equatorial C-2 hydroxyl moiety tofurnish 4.

The ecdysone-inducible gene expression system was employed to assess theability of β-ecdysone 1, and its caged counterpart 4, to induceluciferase expression in the absence and presence of light. We note thatnaturally occurring β-ecdysone homologues, such as muristerone 6 andponasterone A 7 (FIG. 5), are 1,000-times as active as β-ecdysone andinduce transcriptional activity of up to 20,000-fold in cell-basedsystems (No et al., 1996; Albanese et al., 2000). However, since thesehomologues are expensive, we chose to develop our initial chemistry onthe more readily available congener 1. β-ecdysone induces a nearly100-fold enhancement in luciferase activity versus background in 293Tcells that were transiently transfected to constitutively expressEcR/RXR and inducibly express luciferase (Table 1). By contrast, cagedβ-ecdysone 4 is virtually inactive. However, incubation of 293T cellswith 4 and subsequent illumination for 1 min induces luciferaseformation at a level that is nearly 60% of its native bioactivecounterpart 1. The 60% restoration of luciferase activity is consistentwith our observation that a 1 min photolysis time window convertsapproximately 60% of the caged compound 4 into the uncaged β-ecdysone asassessed by HPLC. Various control experiments confirmed that the cagedderivative, in conjunction with light, is sufficient to activate theEcR/RXR expression system (see Results Section).

What is the time-dependence of β-ecdysone-induced luciferase expression?We addressed this question by pre-incubating 293T cells with the cagedderivative 4 for 16 hr followed by illumination for 1 min. Cells weresubsequently incubated for various time intervals, lysed, and luciferaseactivity assessed. Minimal luciferase activity was first observed at the4 hr post-illumination time point and ultimately approached maximalactivity levels at the 16 hr post-illumination time point (FIG. 3).

Is the caged derivative 4 cell permeable and does it undergolight-driven intracellular uncaging? In order to examine these issues,cells were once again incubated with 4 for 16 hr. However, the cellculture was subsequently washed with PBS to remove all extracellular 4.Ecdysone-free media was then added and the cells were immediatelyilluminated for 1 min. Under these conditions, the ecdysteroid shouldonly be present in the intracellular compartment. The cells were thenincubated for various time points, lysed, and the lysate subsequentlytested for luciferase activity. Both sets of conditions(extracellular+intracellular caged β-ecdysone versus intracellular cagedβ-ecdysone only) furnish essentially identical kinetic profiles ofluciferase induction over the first 6 hr following illumination.Thereafter, however, luciferase activity levels off and begins todecrease in the cell culture where caged β-ecdysone was removed from theextracellular milieu. The latter result may be due to the egress ofintracellular β-ecdysone in the absence of an extracellular β-ecdysoneconcentration gradient.

These experiments suggest that 4 is both membrane permeable andintracellularly uncaged following photolysis. However, if photo-uncagedintracellular β-ecdysone does migrate to the extracellular environmentthen it is possible that sites distant from the region of illuminationcould undergo unintentional gene activation as well. On the other hand,given the small intracellular volume of a typical cell (˜1 pL) it islikely that any uncaged β-ecdysone that escapes from its intracellularlocale will be too dilute to effect changes in gene expression profilesat remote sites. We explicitly addressed this possibility by examiningspatially-discrete gene activation using spot illumination of cultured293T cells. Luciferase expression in fixed and permeabilized cells wasvisually identified via the coupled use of a luciferase antibody and asecondary Alexa-labeled antibody. Cells incubated with bioactiveβ-ecdysone 1 for 16 hr furnished global luciferase expression throughoutthe cell culture (FIG. 4 a). Luciferase expression is not observed inthe absence of β-ecdysone (data not shown) nor in the presence of cagedβ-ecdysone without light (see below). By contrast, spatially discreteluciferase expression is observed when 293T cells were incubated withcaged β-ecdysone 4, subsequently washed to remove extracellular (but notintracellular) 4, spot illuminated (˜0.25 mm²) for 10 sec (FIG. 4 b),and returned to the incubator for 6 hr. Regions outside of the zone ofillumination fail to display luciferase expression. These results notonly confirm that caged 4 and light are required to activate geneexpression (FIG. 4 c), but also indicate that a well-defined cellularzone of light-induced protein expression is feasible using thisstrategy.

Caged small molecules (e.g. ATP, NO, etc) have proven to beextraordinarily useful as reagents to help define temporal relationshipsin biochemical-mediated processes (Adams & Tsien, 1993). More recently,caged peptides and proteins have been described, and their use as toolsto delineate the role of individual proteins in cell-based phenomena isunderway (Curley & Lawrence, 1999; Marriott & Walker, 1999). Althoughconcerns have been raised about the effect of light and/or the cagingagent by-product on cell viability we, as well as others, have foundthat light-induced activation of caged compounds (from cagedfluorophores to caged enzymes) has no obvious effect on cell viability.Indeed, cells behave as expected when the bioactive species is generated(see, e.g., the effect of caged cAMP-dependent protein kinase oncellular phenotype, as described by Curley & Lawrence, 1999). Some ofthese concerns arise as a consequence of the relatively longillumination times required for photoactivation in vitro, which aretypically on the order of minutes. However, cell-based experimentsperformed under the microscope require only a few seconds to generatethe uncaged species (due to a high photon flux through a narrow spatialwindow). In addition, although the nitrobenzyl byproduct of the uncagingprocess is an electrophile, extensive work with a wide variety of cagedspecies has failed to uncover any apparent deleterious effects on cellperformance or viability (possibly owing to the high intracellularconcentration of glutathione, which may chemically add to and thereforeneutralize the by-product) (Kaplan, 1978; Walker, 1988).

In summary, we have developed a light-initiated ecdysone-based geneactivation strategy that furnishes both temporal and spatial controlover protein expression in living cells. The ecdysone gene activationsystem is endowed with a number of favorable attributes, including lowbasal activity in the absence of, and high inducibility in the presenceof, the ecdysteroid. Furthermore, ecdysteroids do not appear to have anyeffect on mammalian physiology. These characteristics, in conjunctionwith light-driven spatial control over where expression occurs, providea means to assess the effect and influence of protein expression onphenotype as a function of tissue microenvironment. For example,tumorigenic potential is dependent, in part, on the ability of the tumorto overcome the unique growth suppressive influence associated with thedistinctive microenvironment that harbors it (Roskelley & Bissell,2002). In addition, tumor metastasis is known to be dependent upon thenature of the host microenvironment as well (Liotta & Kohn, 2001).Consequently, the ability to alter gene expression, in amicroenvironment-specific fashion, should prove valuable in assessingthe influence of protein function on the progression of both normal anddisease-related phenomena.

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantages attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

All references cited in this specification are hereby incorporated byreference in their entireties. The discussion of the references hereinis intended merely to summarize the assertions made by the authors andno admission is made that any reference constitutes prior art.Applicants reserve the right to challenge the accuracy and pertinence ofthe cited references.

1. A compound comprising a ligand that specifically reacts with a firstreceptor not naturally present in mammals, wherein the compound furthercomprises a molecular cage covalently bound to the ligand that preventsreaction of the ligand with the first receptor, wherein the ligand isreleased from the cage and capable of reacting with the first receptorupon exposure of the compound to light.
 2. The compound of claim 1,wherein the first receptor is an ecdysone receptor.
 3. The compound ofclaim 1, wherein the ligand is a steroid.
 4. The compound of claim 1,wherein the ligand is an inhibitor of the first receptor.
 5. Thecompound of claim 2, wherein the ligand is selected from the groupconsisting of ecdysone, 20-hydroxyecdysone, ponasterone A, muristeroneA, inokosterone, 3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide and adibenzoylhydrazine.
 6. The compound of claim 1, wherein the molecularcage is a nitromethoxybenzyl moiety.
 7. The compound of claim 6, whereinthe nitromethoxybenzyl moiety is 1-methyl-4,5-dimethoxy-2-nitrobenzene.8. The compound of claim 7, wherein the compound is 4 of FIG.
 1. 9. Thecompound of claim 1, wherein the light comprises wavelengths at 300-400nm.
 10. The compound of claim 9, wherein the light comprises wavelengthsat 325-375 nm.
 11. The compound of claim 1, wherein the molecular cageis a two-photon cage.
 12. A cell of a species, wherein the cell istransfected with a gene of interest and a gene encoding a firstreceptor, the gene of interest operably linked to a genetic elementcapable of being induced by the first receptor when bound to a ligand,and the first receptor not naturally present in the species, the cellfurther comprising a compound comprising the ligand and a molecular cagecovalently bound to the ligand that prevents reaction of the ligand withthe first receptor, wherein the ligand is released from the cage andcapable of reacting with the first receptor upon exposure of thecompound to light.
 13. The cell of claim 12, wherein the cell isprokaryotic.
 14. The cell of claim 12, wherein the cell is eukaryotic.15. The cell of claim 14, wherein the cell is part of a livingmulticellular organism.
 16. The cell of claim 15, wherein substantiallyall of the cells of a cell type in the organism are transfected with thegene of interest and the first receptor.
 17. The cell of claim 15,wherein substantially all of the cells in the organism are transfectedwith the gene of interest and the first receptor.
 18. The cell of claim14, wherein the cell is an animal cell.
 19. The cell of claim 18,wherein the cell is a mammalian cell.
 20. The cell of claim 16, whereinthe cell is a human cell.
 21. The cell of claim 14, wherein the cell isa plant cell.
 22. The cell of claim 12, wherein the first receptor is anecdysone receptor.
 23. The cell of claim 12, wherein the gene ofinterest encodes an untranslated RNA.
 24. The cell of claim 23, whereinthe untranslated RNA is selected from the group consisting of anantisense RNA, an aptamer, and an siRNA.
 25. The cell of claim 12,wherein the gene of interest encodes a protein selected from the groupconsisting of an apoptosis-inducing protein, a protein comprising anapoptosis-inducing protein, a protein comprising an antibody bindingdomain, an angiogenic factor, a cytokine, a viral receptor, a bloodprotein, a transcription factor, a structural protein, a viral protein,a bacterial protein, a recombinase, an extracellular protein, a proteinalready present in the cell, and an engineered protein with no naturalcounterpart.
 26. The cell of claim 12, wherein the gene of interestencodes a Cre recombinase, and wherein the cell further comprises atarget sequence flanked by two loxP sites, wherein the target sequenceis eliminated from the cell when the Cre recombinase is induced.
 27. Thecell of claim 26, wherein the target sequence comprises a promoter, thepromoter operably linked to a target gene.
 28. The cell of claim 27,wherein the target gene is within the target sequence.
 29. The cell ofclaim 26, wherein the target sequence is 3′ from a genetic element, suchthat when the target sequence is eliminated by the Cre recombinase, thegenetic element becomes operably linked to a second gene of interest.30. The cell of claim 29, wherein the second gene of interest encodes anuntranslated RNA.
 31. The cell of claim 30, wherein the untranslated RNAis selected from the group consisting of an antisense RNA, an aptamer,and an siRNA.
 32. The cell of claim 29, wherein the second gene ofinterest is a gene encoding a protein selected from the group consistingof an apoptosis-inducing protein, a protein comprising anapoptosis-inducing protein, a protein comprising an antibody bindingdomain, an angiogenic factor, a cytokine, a blood protein, atranscription factor, a structural protein, a viral protein, a bacterialprotein, a viral receptor, an extracellular protein, a protein alreadypresent in the cell, and an engineered protein with no naturalcounterpart.
 33. The cell of claim 27, wherein the promoter isinducible.
 34. The cell of claim 27, wherein the promoter isconstitutive.
 35. The cell of claim 25, wherein the target sequence is atarget gene.
 36. The cell of claim 35, wherein the target gene encodesan untranslated RNA.
 37. The cell of claim 36, wherein the untranslatedRNA is selected from the group consisting of an antisense RNA, anaptamer, and an siRNA.
 38. The cell of claim 35, wherein the target geneis a gene encoding a protein selected from the group consisting of anapoptosis-inducing protein, a protein comprising an antibody bindingdomain, an angiogenic factor, a cytokine, a blood protein, atranscription factor, a structural protein, a viral protein, a bacterialprotein, a viral protein, an extracellular protein, a protein alreadypresent in the cell, and an engineered protein with no naturalcounterpart.
 39. The cell of claim 26, wherein the target sequencecomprises a stop codon.
 40. The cell of claim 12, wherein the gene ofinterest encodes a viral receptor, the viral receptor allowing entry ofa viral vector into the cell.
 41. The cell of claim 40, wherein theviral receptor is a TVA receptor for subgroup A avian leucosis virus andthe viral vector is a subgroup A avian leucosis virus vector.
 42. Thecell of claim 40, wherein the cell has been exposed to light and a viralvector expressing a second gene of interest, wherein the viral vectorhas infected the cell and expresses the second gene of interest.
 43. Thecell of claim 42, wherein the second gene of interest is a gene encodinga protein selected from the group consisting of an apoptosis-inducingprotein, a protein comprising an apoptosis-inducing protein, a proteincomprising an antibody binding domain, an angiogenic factor, a cytokine,a blood protein, a transcription factor, a structural protein, a viralprotein, a bacterial protein, an extracellular protein, a proteinalready present in the cell, and an engineered protein with no naturalcounterpart.
 44. The cell of claim 42, wherein the second gene ofinterest encodes an untranslated RNA.
 45. The cell of claim 44, whereinthe untranslated RNA is selected from the group consisting of anantisense RNA, an aptamer, and an siRNA.
 46. The cell of claim 12,wherein the compound comprises a ligand of an ecdysone receptor.
 47. Thecell of claim 12, wherein the light comprises wavelengths at 325-375 nm.48. The cell of claim 12, wherein the light comprises wavelengths atabout 700 nm.
 49. The cell of claim 12, wherein the molecular cage is anitromethoxybenzyl moiety.
 50. The cell of claim 49, wherein thenitromethoxybenzyl moiety is 1-methyl-4,5-dimethoxy-2-nitrobenzene. 51.The cell of claim 49, wherein the molecular cage is a two-photon cage.52. The cell of claim 12, wherein the gene of interest and the geneencoding a first receptor are transfected into the cell with a viralvector.
 53. The cell of claim 12, wherein the gene of interest and thegene encoding a first receptor are transfected into the cell with aplasmid vector.
 54. The cell of claim 12, wherein the gene of interestand the gene encoding a first receptor are transiently expressed. 55.The cell of claim 12, wherein the gene of interest and the gene encodinga first receptor are stably expressed.
 56. The cell of claim 12, whereinthe gene of interest and the gene encoding a first receptor aremaintained extrachromosomally in the cell.
 57. The cell of claim 12,wherein the gene of interest and the gene encoding a first receptor areintegrated into a chromosome of the cell.
 58. A method of expressing agene of interest in a cell of a species, the method comprising creatingthe cell of a species of claim 12 by transfecting the cell with the geneof interest and a gene encoding a first receptor, the gene of interestoperably linked to a genetic element capable of being induced by thefirst receptor when bound to a ligand, the first receptor not naturallypresent in the species; and adding a compound to the cell, the compoundcomprising the ligand and a molecular cage covalently bound to theligand that prevents reaction of the ligand with the first receptor, theligand capable of being released from the cage upon exposure of thecompound to light; then exposing the cell to light sufficient to releasethe cage from the ligand.
 59. The method of claim 58, wherein the cellis part of a living multicellular organism.
 60. The method of claim 59,wherein substantially all of the cells of a cell type in the organismare transfected with the gene of interest and the first receptor. 61.The method of claim 59, wherein substantially all of the cells in theorganism are transfected with the gene of interest and the firstreceptor.
 62. The method of claim 58, wherein the light compriseswavelengths at 300-400 nm.
 63. The method of claim 59, wherein the lightcomprises wavelengths at 325-375 nm.
 64. The method of claim 58, whereinthe light comprises wavelengths at about 700 nm.
 65. The method of claim58, wherein the gene of interest and the gene encoding a first receptorare transfected into the cell with a viral vector.
 66. The method ofclaim 58, wherein the gene of interest and the gene encoding a firstreceptor are transfected into the cell with a plasmid vector.
 67. Amethod of expressing a second gene of interest in a cell of a species,the method comprising transfecting the cell with a first gene ofinterest and a gene encoding a first receptor, the first gene ofinterest encoding a viral receptor, the viral receptor allowing entry ofa viral vector into the cell, the first gene of interest operably linkedto a genetic element capable of being induced by the first receptor whenbound to a ligand, the first receptor not naturally present in thespecies, the ligand further comprising a molecular cage covalently boundto the ligand that prevents reaction of the ligand with the firstreceptor, wherein the ligand is released from the cage and capable ofreacting with the first receptor upon exposure of the compound to light;exposing the cell to the viral vector further comprising an expressiblegene encoding the second gene of interest; then exposing the cell tolight sufficient to release the cage from the ligand, allowing theligand to react with the first receptor, directing expression of theviral receptor and allowing infection of the cell by the viral vector;then expressing the second gene of interest.
 68. The method of claim 67,wherein the viral receptor is a TVA receptor for subgroup A avianleucosis virus and the viral vector is a subgroup A avian leucosisvirus.
 69. The method of claim 67, wherein the first receptor is anecdysone receptor.
 70. The method of claim 67, wherein the ligand is asteroid.
 71. The method of claim 69, wherein the ligand is selected fromthe group consisting of ecdysone, 20-hydroxyecdysone, ponasterone A,muristerone A, inokosterone,3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide and adibenzoylhydrazine.
 72. The method of claim 67, wherein the second geneof interest is operably linked to an inducible promoter.
 73. The methodof claim 67, wherein the second gene of interest is operably linked to aconstitutive promoter.
 74. The method of claim 67, wherein the secondgene of interest is a gene encoding a protein selected from the groupconsisting of an apoptosis-inducing protein, a protein comprising anapoptosis-inducing protein, a protein comprising an antibody bindingdomain, an angiogenic factor, a cytokine, a blood protein, atranscription factor, a structural protein, a viral protein, a bacterialprotein, an extracellular protein, a protein already present in thecell, and an engineered protein with no natural counterpart.
 75. Themethod of claim 67, wherein the second gene of interest encodes anuntranslated RNA.
 76. The method of claim 75, wherein the untranslatedRNA is selected from the group consisting of an antisense RNA, anaptamer, and an siRNA.
 77. A method of repressing a gene of interest ina cell of a species, the method comprising transfecting the cell withthe gene of interest and a gene encoding a first receptor, the gene ofinterest operably linked to a genetic element capable of being repressedby the first receptor when bound to a ligand; adding a compound to thecell, the compound comprising the ligand and a molecular cage covalentlybound to the ligand that prevents reaction of the ligand with the firstreceptor, the ligand capable of being released from the cage uponexposure of the compound to light; then exposing the cell to lightsufficient to release the cage from the ligand.
 78. The method of claim77, wherein the first receptor is a transcriptional co-repressor. 79.The method of claim 77, wherein the first receptor is not naturallypresent in the species.
 80. The method of claim 77, wherein the cell isprokaryotic.
 81. The method of claim 77, wherein the cell is eukaryotic.82. The method of claim 81, wherein the cell is part of a livingmulticellular organism.
 83. The method of claim 82, whereinsubstantially all of the cells of a cell type in the organism aretransfected with the gene of interest and the gene encoding a firstreceptor.
 84. The method of claim 82, wherein substantially all of thecells in the organism are transfected with the gene of interest and thefirst receptor.
 85. The method of claims 81, wherein the cell is ananimal cell.
 86. The method of claim 85, wherein the cell is a mammaliancell.
 87. The method of claim 85, wherein the cell is a human cell. 88.The method of claim 81, wherein the cell is a plant cell.
 89. The methodof claim 77, wherein the gene of interest encodes an untranslated RNA.90. The method of claim 89, wherein the untranslated RNA is selectedfrom the group consisting of an antisense RNA, an aptamer, and an siRNA.91. The method of claim 77, wherein the gene of interest encodes aprotein selected from the group consisting of an apoptosis-inducingprotein, a protein comprising an antibody binding domain, an angiogenicfactor, a cytokine, a blood protein, a viral receptor, a transcriptionfactor, a structural protein, a viral protein, a bacterial protein, anextracellular protein, a protein already present in the cell, and anengineered protein with no natural counterpart.
 92. The method of claim77, wherein the light comprises wavelengths at 300-400 nm.
 93. Themethod of claim 77, wherein the light comprises wavelengths at 325-375nm.
 94. The method of claim 77, wherein the molecular cage is anitromethoxybenzyl moiety.
 95. The method of claim 94, wherein thenitromethoxybenzyl moiety is 1-methyl-4,5-dimethoxy-2-nitrobenzene. 96.The method of claim 77, wherein the molecular cage is a two-photon cage.97. The method of claim 77, wherein the gene of interest and the geneencoding a first receptor are transfected into the cell with a viralvector.
 98. The method of claim 77, wherein the gene of interest and thegene encoding a first receptor are transfected into the cell with aplasmid vector.
 99. The method of claim 77, wherein the gene of interestand the gene encoding a first receptor are transiently expressed. 100.The method of claim 77, wherein the gene of interest and the geneencoding a first receptor are stably expressed.
 101. The method of claim77, wherein the gene of interest and the gene encoding a first receptorare maintained extrachromosomally in the cell.
 102. The method of claim77, wherein the gene of interest and the gene encoding a first receptorare integrated into a chromosome of the cell.
 103. A method of inducingelimination of a target sequence in a cell of a species, the methodcomprising transfecting the cell with a gene encoding a recombinaseoperably linked to a genetic element capable of being induced by a firstreceptor when bound to a ligand, wherein the first receptor is capableof inducing the genetic element when the first receptor reacts with aligand; and a gene encoding the first receptor; adding a compound to thecell, the compound comprising the ligand and a molecular cage covalentlybound to the ligand that prevents reaction of the ligand with the firstreceptor, the ligand capable of being released from the cage uponexposure of the compound to light; and subsequently exposing the cell tolight sufficient to release the cage from the ligand.
 104. The method ofclaim 103, wherein the recombinase is a Cre recombinase, and wherein thecell is also transfected with two loxP sites flanking the targetsequence.
 105. The method of claim 103, wherein the first receptor isnot naturally present in the species.
 106. The method of claim 103,wherein the compound comprises a ligand that specifically reacts with afirst receptor not naturally present in mammals, wherein the compoundfurther comprises a molecular cage covalently bound to the ligand thatprevents reaction of the ligand with the first receptor, wherein theligand is released from the cage and capable of reacting with the firstreceptor upon exposure of the compound to light.
 107. The method ofclaim 103, wherein the cell is prokaryotic.
 108. The method of claim103, wherein the cell is eukaryotic.
 109. The method of claim 108,wherein the cell is part of a living multicellular organism.
 110. Themethod of claim 109, wherein substantially all of the cells of a celltype in the organism are transfected with the gene of interest and thefirst receptor.
 111. The method of claim 109, wherein substantially allof the cells in the organism are transfected with the gene of interestand the first receptor.
 112. The method of claim 108, wherein the cellis an animal cell.
 113. The method of claim 112, wherein the cell is amammalian cell.
 114. The method of claim 112, wherein the cell is ahuman cell.
 115. The method of claim 108, wherein the cell is a plantcell.
 116. The method of claim 103, wherein the target sequence encodesan untranslated RNA.
 117. The method of claim 116, wherein theuntranslated RNA is selected from the group consisting of an antisenseRNA, an aptamer, and an siRNA.
 118. The method of claim 103, wherein thetarget sequence encodes a protein selected from the group consisting ofan apoptosis-inducing protein, a protein comprising an antibody bindingdomain, an angiogenic factor, a cytokine, a blood protein, a viralreceptor, a transcription factor, a structural protein, a viral protein,a bacterial protein, an extracellular protein, a protein already presentin the cell, and an engineered protein with no natural counterpart. 119.The method of claim 103, wherein the target sequence encodes a promoter.120. The method of claim 103, wherein the elimination of the targetsequence brings a promoter adjacent to a gene of interest such that thepromoter is operably linked to the gene of interest.
 121. The method ofclaim 120, wherein the target sequence encodes an untranslated RNA. 122.The method of claim 121, wherein the untranslated RNA is selected fromthe group consisting of an antisense RNA, an aptamer, and an siRNA. 123.The method of claim 120, wherein the target sequence encodes a proteinselected from the group consisting of an apoptosis-inducing protein, aprotein comprising an antibody binding domain, an angiogenic factor, acytokine, a blood protein, a transcription factor, a structural protein,a viral protein, a bacterial protein, a viral receptor, an extracellularprotein, a protein already present in the cell, and an engineeredprotein with no natural counterpart.
 124. The method of claim 103,wherein the target sequence comprises a stop codon.
 125. The method ofclaim 103, wherein the gene encoding a Cre recombinase, the two loxPsites, and the gene encoding a first receptor are transfected into thecell with a viral vector.
 126. The method of claim 103, wherein the geneencoding a Cre recombinase, the two loxP sites, and the gene encoding afirst receptor are transfected into the cell with a plasmid vector. 127.The method of claim 103, wherein the gene encoding a Cre recombinase,the two loxP sites, and the gene encoding a first receptor aretransiently expressed.
 128. The method of claim 103, wherein the geneencoding a Cre recombinase, the two loxP sites, and the gene encoding afirst receptor are stably expressed.
 129. The method of claim 103,wherein the gene encoding a Cre recombinase, the two loxP sites, and thegene encoding a first receptor are maintained extrachromosomally in thecell.
 130. The method of claims 103, wherein the gene encoding a Crerecombinase, the two loxP sites, and the gene encoding a first receptorare integrated into a chromosome of the cell.
 131. A kit for theconditional expression of a gene of interest in a cell, the kitcomprising, in suitable containers, the compound of claim 1 and a vectorcomprising a gene encoding the first receptor.
 132. The kit of claim131, further comprising a first vector comprising a gene encoding aviral receptor, the viral receptor allowing entry of a viral vector intoa cell, and the viral vector comprising a site for insertion of the geneof interest such that the gene of interest can be expressed when theviral vector infects the cell.
 133. The kit of claim 132, wherein theviral receptor is a TVA receptor for subgroup A avian leucosis virus andthe viral vector is a subgroup A avian leucosis virus.
 134. The kit ofclaim 132, wherein the site for insertion of the gene of interest isoperably linked to an inducible promoter.
 135. The kit of claim 132,wherein the site for insertion of the gene of interest is operablylinked to a constitutive promoter.
 136. The kit of claim 131, whereinthe cell is a mammalian cell.
 137. The kit of claim 131, furthercomprising instructions for expressing the gene of interest in the celltransfected with the vector, and exposed to the compound and light. 138.A kit for the conditional elimination of a target sequence in a cell,the kit comprising, in suitable containers, one or more vectorscomprising a gene encoding a recombinase operably linked to a geneticelement capable of being induced by a first receptor when bound to aligand, wherein the first receptor is capable of inducing the geneticelement when the first receptor reacts with a ligand; a gene encodingthe first receptor; and a compound comprising the ligand and a molecularcage covalently bound to the ligand that prevents reaction of the ligandwith the first receptor, wherein the ligand is released from the cageand capable of reacting with the first receptor upon exposure of thecompound to light.
 139. The kit of claim 138, wherein the recombinase isa Cre recombinase.
 140. The kit of claim 138, wherein the first receptoris an ecdysone receptor.
 141. The kit of claim 138, wherein the ligandis a steroid.
 142. The kit of claim 140, wherein the ligand is selectedfrom the group consisting of ecdysone, 20-hydroxyecdysone, ponasteroneA, muristerone A, inokosterone,3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide and adibenzoylhydrazine.
 143. The kit of claim 138, further comprisinginstructions for using the kit to eliminate a target sequence in cellstransfected with the vectors and exposed to the compound and to light.